DESCRIPTION: The long term goal of this research is to elucidate the mechanism and regulation of transcription in eukaryotes. Dr. Kornberg will focus on RNA polymerase II, the enzyme responsible for almost all eukaryotic transcription, whose intricate regulation underlies many cellular and developmental processes. Most regulation occurs at the initiation of transcription, which involves the interaction of RNA polymerase II with a set of general transcription factors at a promoter. RNA polymerase II is itself a large assembly: the yeast enzyme is made up of 15 polypeptide chains, with a mass of nearly 600,000 Daltons. The entire initiation complex comprises more than 25 polypeptides, with a total mass in excess of 1.2 million Daltons. Dr. Kornberg wishes to determine the three-dimensional structure of the initiation complex as an essential step towards understanding the mechanism and regulation of transcription. Dr. Kornberg will accomplish this objective in stages: the x-ray structures of the polymerase and general transcription factors will be determined individually and then combined on the basis of structures of complexes revealed by electron microscope crystallography. Dr. Kornberg proposes here the x-ray analysis of RNA polymerase II crystals, and the formation and analysis of cocrystals of the polymerase with DNA, RNA, and general initiation factors. The results should apply to RNA polymerases and transcription mechanisms in general, since the polymerases, transcription factors, and aspects of the mechanism and regulation of transcription have been conserved through evolution. Aims and experimental approaches of the proposed research include the following: the x-ray structure of yeast RNA polymerase II will be determined first at low (6 Angstrom) and then at atomic resolution; cocrystals of RNA polymerase II with DNA and RNA oligonucleotides will be analyzed by difference Fourier methods; and cocrystals of RNA polymerase II with general transcription factors, including TFIIB and TFIIE, will be prepared and subjected to structural study as well.